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WO2011029013A2 - Obtention de produits biologiques secrétés par microbes photosynthétiques - Google Patents

Obtention de produits biologiques secrétés par microbes photosynthétiques Download PDF

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WO2011029013A2
WO2011029013A2 PCT/US2010/047830 US2010047830W WO2011029013A2 WO 2011029013 A2 WO2011029013 A2 WO 2011029013A2 US 2010047830 W US2010047830 W US 2010047830W WO 2011029013 A2 WO2011029013 A2 WO 2011029013A2
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photosynthetic bacterium
transporter
gene
recombinant
recombinant photosynthetic
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WO2011029013A3 (fr
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Jeffrey Charles Way
Henrike Niederholtmeyer
Bernd Wolfstaedter
David Savage
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Harvard University
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Harvard University
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Priority to EP10814563.2A priority Critical patent/EP2473597A4/fr
Priority to US13/394,070 priority patent/US20120220007A1/en
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Publication of WO2011029013A3 publication Critical patent/WO2011029013A3/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0008—Oxidoreductases (1.) acting on the aldehyde or oxo group of donors (1.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1294—Phosphotransferases with paired acceptors (2.7.9)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/14—Glutamic acid; Glutamine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/02—Monosaccharides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/56—Lactic acid

Definitions

  • the invention relates to compositions and methods for the production of useful chemicals from photosynthetic microbes.
  • photosynthetic microbes could directly produce such molecules, much as ethanol is produced by yeast, because of the efficient use of light energy by these organisms and the potential for C0 2 mitigation during production.
  • ethanol is produced by yeast
  • most photosynthetic bacteria do not secrete carbon-based molecules, likely because of the metabolic cost of their synthesis.
  • hydrophilic molecules such as glucose, lactic acid and succinic acid cannot pass through cell membranes under normal conditions. Therefore, there is a need in the art for methods and systems that allow the production and secretion of hydrophilic molecules from photosynthetic bacteria.
  • the present invention provides compositions and methods for the production of useful chemicals from photosynthetic bacteria, such as cyanobacteria.
  • the invention provides genetically engineered photo synthetic cyanobacteria that express at least one heterologous enzyme and at least one heterologous transporter, wherein the transporter mediates the secretion of a product into an extracellular medium.
  • the transporter mediates the secretion of a product whose synthesis is enhanced by the heterologous enzyme.
  • the heterologous enzyme is an invertase, such as invA; and the transporter is glf.
  • the heterologous enzyme is a dehydrogenase, such as IdhA, and the transporter is lldP.
  • the cyanobacteria may further include a resistance marker.
  • the heterologous transporter is a member of the major facilitator superfamily, including the Glut family of glucose transport proteins, the HXT family of glucose transporters, lacY, lldP, a mammalian citric acid transport protein, the glpT glycerol phosphate transporter, a sucrose transporter, an amino acid transporter, a glutamate transporter, and GLF.
  • the heterologous hexose (e.g., glucose) transporters can be the Glut, HXT, and GLF proteins.
  • the recombinant photo synthetic microbes further comprise additional heterologous genes that encode enzymes for enhancing synthesis of intracellular precursors.
  • the additional heterologous gene is udhA, which encodes NAD(P)H transhydrogenase.
  • the additional heterologous gene is udhA, which encodes NAD(P)H transhydrogenase.
  • heterologous gene is galU, which encodes UDP-glucose phosphorylase.
  • the additional heterologous gene encodes an enzyme for sucrose synthesis, such as sucrose phosphate synthase and sucrose phosphate phosphatase.
  • the recombinant microbe further comprises at least one reduction-of-function mutation that can enhance production of hydrophilic molecules.
  • the reduction-of-function mutation is a deletion in at least one endogenous kinase gene that phosphorylates a hydrophilic product.
  • the endogenous kinase is hexokinase.
  • the reduction-of-function mutation is a deletion in genes encoding any membrane- associated NAD(P) transhydrogenase.
  • genetically engineered photosynthetic microbes are cyanobacteria.
  • the transporter is a member of the major facilitator superfamily.
  • the compound is a chiral compound. Examples of chiral compounds include, without limitation, sugars and carboxylic acids, such as lactic acid.
  • sugars include glucose, fructose, a mixture of glucose and fructose, or sucrose.
  • the enzyme is expressed from an inducible promoter.
  • inducible promoters include a lac operon promoter, a nitrogen- sensitive promoter, and a salt-inducible promoter.
  • the inducible promoter is IPTG-inducible promoter.
  • the inducible promoter is NaCl- inducible promoter.
  • the invention provides for a recombinant photosynthetic bacterium, such as cyanobacterium, that expresses at least one heterologous transporter that is coupled to the pH gradient across a cell membrane.
  • the invention provides for a recombinant photosynthetic bacterium, such as a cyanobacterium, that expresses at least one heterologous transporter that co-transports a proton with a product (e.g., a metabolite).
  • the invention provides genetically engineered photosynthetic bacteria, such as cyanobacteria, that express a heterologous transporter that co- transports a sodium ion with a product (e.g., a metabolite).
  • a product e.g., a metabolite
  • the heterologous transporter mediates the symport or antiport of a product and an inorganic ion.
  • an inorganic ion include a proton, a sodium ion, a potassium ion, a chloride ion, and a hydroxide ion.
  • the heterologous transporter can be a lactic acid/proton symporter, a sucrose proton symporter, a lactose/proton symporter, a dicarboxylic acid/proton symporter, an amino acid/proton symporter, a citrate/proton symporter, an amino acid/sodium ion symporter, or an amino acid/hydroxide ion antiporter.
  • heterologous transporters include, but not limited to, a sucrose transporter CscB, a lactose permease of E. coli encoded by the lacY gene, a mammalian H + /peptide transporter PepTlH, a glutamate transporter GltS, and a lactate transporter lldP.
  • the invention provides a method for producing a hydrophilic product, the method comprising culturing a recombinant cyanobacterium according to the invention in the presence of light and C0 2 , and obtaining the product from a supernatant of the cultured microbe.
  • the method further includes providing an inducing agent to induce the expression of at least one heterologous gene expressed in the recombinant bacterium.
  • the heterologous gene encodes an enzyme, a transporter or both.
  • the hydrophilic product is glucose and/or fructose, or lactic acid.
  • the invention provides a method for producing a hydrophilic product, the method comprising culturing a cyanobacteria according to the second aspect of the invention in the presence of light and C0 2 , and obtaining said product from a supernatant of the cultured microbe.
  • the method further includes providing an inducing agent to induce the expression of at least one heterologous gene expressed in cyanobacteria.
  • the heterologous gene encodes an enzyme, a transporter or both.
  • the hydrophilic product is lactic acid, sucrose, an amino acid, citric acid, malic acid, fumaric acid, or succinic acid.
  • Figures 1A and IB show schematic diagrams of engineering strategies for production of bioproducts by a genetically engineered cyanobacterium.
  • Figure 1A illustrates a cyanobacterial cell in which one or more heterologous enzymes have been expressed to produce a hydrophilic metabolite that cannot spontaneously cross a cell membrane, and in which a heterologous transporter has been introduced to allow secretion of the metabolite from the cell.
  • Figure IB shows an engineering scheme for production of hexose sugars (e.g., glucose and fructose) by a genetically engineered Synechococcus elongatus 7942. Synechococcus naturally produces sucrose in response to salt stress by fixing carbon via natural pathways such as Calvin Cycle.
  • hexose sugars e.g., glucose and fructose
  • the bacteria are engineered to express invertase (encoded by invA), which cleaves sucrose into glucose and fructose, and the glf gene encoding a glucose- and fructose-facilitated diffusion transporter, which allows export of the sugars from the cell.
  • invertase encoded by invA
  • glf a glucose- and fructose-facilitated diffusion transporter
  • Figure 2 shows plasmid vectors that mediate integration of the invA and glf genes into the S. elongatus 7942 genome by homologous recombination. Derivatives of S. elongatus were constructed by insertion of transgenes into "neutral sites" NS1 and NS2 with
  • Figures 3A and 3B show the gene and protein sequences of the inserted invA into S. elongatus 7942.
  • Figure 3A shows the sequence of invA, spectinomycin-resistance, and lad genes inserted into NS1 of S. elongatus 7942. The region encoding the invA gene plus immediately flanking regions is shown in capital letters. This gene is encoded in the antisense orientation. The start and stop codons are underlined.
  • the plasmid carrying the invA gene is termed DS1321.
  • Figure 3B shows the sequence of the invertase protein, including a His 6 tag at the C-terminus, as expressed in S. elongatus 7942.
  • Figures 4A and 4B show the gene and protein sequences of the inserted glf into S. elongates 7942.
  • Figure 4A shows the sequence of glf, kanamycin-resistance, and lad genes inserted into NS2 of S. elongatus 7942. The region encoding g//and immediately flanking sequences are shown in capitals, with the start and stop codons underlined.
  • the plasmid carrying the glf gene is termed DS21.
  • Figure 4B shows the sequence of the GLF protein, including a His6 tag at the C-terminus, as expressed in S. elongatus 7942.
  • Figures 5A to 5D show the expression of transgenes to promote hexose sugar or lactate synthesis and secretion.
  • Figure 5A shows the western blot assay for expression of His 6 - tagged invA and glf in E. coli and Synechococcus. Predicted molecular weights of tagged InvA and GLF are 60,000 and 55,400 respectively. Symbols '-' and '+' indicate samples growth without or with 1 mM IPTG. Lane 1 - E. coli, invA expression plasmid; lane 2 - E. coli, glf expression plasmid; lane 3 - S.
  • Figure 5B shows a western blot assay for expression of His 6 -tagged IdhA and lldP in Synechococcus containing the IdhA and lldP expression constructs. Lanes 1 to 3: 100 mM, 1 mM, and 0 IPTG, respectively. (The lldP product was not detected.)
  • Figure 5C shows invertase enzyme activity from extracts of
  • FIG. 5D shows functional expression of the glf transporter in Synechococcus, assayed by growth of Synechococcus in the dark.
  • Solid lines growth; dotted lines: disappearance of glucose from culture medium. The lines that remain fairly constant correspond to wild-type S. elongatus 7942; the decreasing dotted line and increasing solid line correspond to recombinant S. elongatus 7942 that expresses the glf gene.
  • Figures 6A and 6B show sugar production in culture medium of Synechococcus strains expressing Z. mobilis glf and invA (cross), only glf (triangle), only invA (square), or neither transgene (diamond).
  • the x axis shows the time after induction of sucrose synthesis with 200 mM NaCl and of transgenes with 100 ⁇ IPTG.
  • Symbol "21” refers to plasmid DS21; "13” refers to plasmid DS1321.
  • Figure 6A shows extracellular concentration of glucose of the four strains.
  • Figure 6B shows extracellular concentration of fructose of the four strains.
  • Figures 7 shows the growth curves of cyanobacteria engineered to express invA and glf from Z. mobilis (square), only glf (cross), only invA (triangle), or neither transgene (diamond).
  • the x axis shows the time after induction of sucrose synthesis with 200 mM NaCl and of transgenes with 100 ⁇ IPTG.
  • Figures 8 shows the extracellular concentration of sucrose produced by
  • Synechococcus strains expressing Z. mobilis glf and invA (glf+invA), only glf (13+glf), only invA (21+invA), or neither transgene (21+13).
  • the bars show the concentrations of both intracellular and extracellular sucrose determined from a Day-3 whole culture sample (cells and culture supernatant); less than 3 ⁇ sucrose was detected in the mvA-expressing strains.
  • Figures 9A and 9B show growth and sugar production or consumption as a function of a cycle consisting of 12-h days and 12-h nights.
  • the lines indicate OD 750 ; bars indicate the concentrations of extracellular fructose (gray bars) and glucose (striped bars).
  • the arrows indicate the induction with NaCl and IPTG at dawn ( Figure 9A) and at dusk ( Figure 9B).
  • FigureslOA to IOC show the growth and sugar production of g//+mvA-expressing and control cells as a function of NaCl concentration.
  • Figure 10A shows the growth rates of a Synechococcus empty vector control. Symbol “21+13" and glf+invA strain after induction with 100 mM IPTG and various different NaCl concentrations in BG-11 medium.
  • Figure 10B shows the concentrations of glucose and fructose in the culture medium of a Synechococcus glf+invA strain four days after induction with 100 mM IPTG and various different NaCl concentrations in BG-11 medium.
  • Figure IOC shows the extracellular concentrations of glucose and fructose produced by a Synechococcus glf+invA strain on a per cell basis.
  • Figures 11A to HE show that sugar-secreting Synechococcus supports E. coli growth in coculture.
  • E. coli DH5oc containing a YFP expression plasmid was diluted to obtain a concentration of 10 6 cells/ml in wild-type Synechococcus cultures or Synechococcus cultures expressing g//and invA in BG-11 medium with 200 mM NaCl, 1 mg/ml NH 4 C1, and appropriate antibiotics.
  • Figure 11A shows continued growth of engineered and wild-type Synechococcus in the presence of E. coli.
  • Figures 11B to 11C show growth of E.
  • Figures 11D to HE show coculture of sugar-secreting Synechococcus and E. coli on agar plates at two different NaCl concentrations.
  • E. coli YFP fluorescence (lighter shade); Synechococcus chlorophyll autofluorescence (coiled features).
  • Figures 12A to 12C show plasmid schematics for expression of IdhA and lldP in S. elongatus 7942.
  • Figure 12A shows the plasmid for expression of IdhA.
  • Figure 12B shows the plasmid for expression of lldP.
  • Figure 12C shows construction of derivatives of S. elongatus by insertion of lldP and IdhA genes into "neutral sites" NSl and NS2 with spectinomycin-resistance and kanamycin-resistance markers, respectively. Strains lacking a particular transgene were transformed with empty spectinomycin-resistant and kanamycin-resistant vectors as controls.
  • Figures 13A to 13B show the sequence of the inserted IdhA gene and expressed protein in S. elongatus 7942.
  • Figure 13A shows the nucleotide sequence of the inserted IdhA gene in S. elongatus. Start and stop codons are underlined.
  • Figure 13B shows the amino acid sequence of IdhA gene encoding lactate dehydrogenase, including a His 6 tag at the N-terminus, as expressed in S. elongatus 7942.
  • Figures 14A and 14B show the sequence of the inserted lldP gene and expressed protein in S. elongatus 7942.
  • Figure 14A shows the nucleotide sequence of the inserted lldP gene in S. elongatus. Start and stop codons are underlined.
  • Figure 14B shows the amino acid sequence of the expressed protein encoded by lldP, including a His 6 tag at the N-terminus, as expressed in S. elongatus 7942.
  • Figures 15A to 15C show schematic diagrams for production of bioproducts by a recombinant cyanobacterium that expresses an ion-coupled transporter.
  • Figure 15A illustrates a cyanobacterial cell in which an ion-coupled transporter is expressed to allow secretion of the product from the cell in a manner that is assisted by the concentration gradient of the ion.
  • FIG 15B shows the engineering scheme for cyanobacterial production of lactic acid (lactate).
  • lactate lactate dehydrogenase
  • lldP transporter
  • Synechococcus naturally produces intracellular sucrose in response to salt stress.
  • the bacteria are engineered to express the cscB gene, which encodes a proton-coupled sucrose transporter.
  • Figures 16A and 16B show lactate production and growth of Synechococcus strains Synechococcus strains expressing E. coli IdhA and lldP (cross), only lldP (triangle), only IdhA (square), or neither transgene (diamond).
  • Figure 16A shows the concentration of secreted lactic acid in culture medium of these four strains.
  • Figure 16B shows the growth of these four strains.
  • Figures 17 A to 17C show rational metabolic engineering of Synechococcus to enhance lactate production.
  • Figure 17A shows a schematic depiction of a model for
  • NADPH is the major carrier of reducing equivalents in photosynthetic microbes. Exchange with NAD + is catalyzed by NADP/NAD transhydrogenase to yield NADH, the reducing agent substrate for pyruvate dehydrogenase.
  • Figure 17B shows lactate
  • FIG. 17C shows the insertion of udhA into "neutral site" NS3 with a chloramphenicol- resistance marker.
  • Figures 18A and 18B depict a new vector, pHNl-LacUV5, for integration of transgenes into the S. elongatus 7942 genome.
  • This vector allows integration at the "NS3" site, that corresponds to a sequence within the remnant of a cryptic prophage, such that integrations are expected to have no phenotypic consequences related to integration per se.
  • Figure 18A shows sites of NS3, transcription terminators, lac operon promoter and multiple cloning sites for genes to be expressed.
  • the sequence of pHNl-LacUV5 vector in Figure 18B includes the origin of replication from pUC57 (bases 6 to 625); an "NS3-II" segment from bases 655 to 1554; a segment containing the E.
  • coli rrnB Tl and T2 terminators from bases 1567 to 1769; the /acUV5 promoter from bases 1791 to 1881; a multiple cloning site including unique Ndel, Xbal, Hindlll, Notl and BamHI sites from bases 1900 to 1934; a coding sequence of a chloramphenicol resistance gene from bases 2037 to 2696; an E. coli lad coding sequence from bases 2839 to 3921; an E. coli Trp operon terminator from bases 3922 to 3972; and an "NS3-I" segment from bases 3977 to 4876.
  • Analogous plasmids that use the weaker wild-type (not UV5) lac promoter and the stronger Trp(-35) lacUV5(-10) promoter were also constructed.
  • Figures 19A to 19C show the enhancement of hexose sugar production by rational metabolic engineering of Synechococcus.
  • Figure 19A shows the rationale for
  • UDP-glucose and fructose-6-P are the precursors of glucose and fructose in the artificial pathway for hexose production.
  • UDP-glucose is produced by UDP-glucose phosphorylase, encoded by the galU gene.
  • Figure 19B shows the total glucose plus fructose concentrations in culture supernatants of induced Synechococcus glf+invA either with or without a galU expression construct. Dashed lines: glucose+fructose concentrations; solid lines: bacterial density.
  • Figure 19C shows the insertion of galU into "neutral site" NS3 with a chloramphenicol-resistance marker.
  • Figures 20A and 20B show the sequence for the CscB protein of E. coli.
  • Figure 20A shows the nucleic acid sequence and Figure 20B shows the encoded amino acids.
  • Figures 21A and 21B show the sequence for the GltS protein of E. coli.
  • Figure 21 A shows the nucleic acid sequence and Figure 21 B shows the corresponding amino acids.
  • the invention provides compositions and methods for the production of useful chemicals from photosynthetic microbes. It is an object of the invention to engineer
  • photosynthetic microbes have evolved to use only C0 2 , fixed or atmospheric nitrogen, and various minerals. Photosynthetic microbes are ⁇ 1 order of magnitude more productive than conventional terrestrial plants to capture solar energy and their
  • photosynthetic efficiencies can be >10%. Huntley et al., 12 Mitigat. Adapt. Strat. Global Change 573 (2007); Li et al., 24 Biotech. Prog. 815 (2008).
  • This self-sufficient mode of metabolism contrasts with that of E. coli, for example, which has a wide variety of transporters.
  • Another point of contrast is that E. coli and many other microbes secrete organic compounds as waste products. Because of the high energetic cost of fixing carbon, photosynthetic microbes generally do not secrete carbon-based waste products.
  • the present invention provides for photosynthetic microbes that are engineered to do so, including cyanobacteria, green sulfur bacteria (including Family Chlowbiaceae), purple sulfur bacteria (e.g., Family Chromatiaceae and Family Ectothiorhodospiraceae) purple nonsulfur bacteria (e.g., Family Rhodospirillaceae), and green bacteria (including Family Chloroflexaceae).
  • cyanobacteria including Green sulfur bacteria (including Family Chlowbiaceae), purple sulfur bacteria (e.g., Family Chromatiaceae and Family Ectothiorhodospiraceae) purple nonsulfur bacteria (e.g., Family Rhodospirillaceae), and green bacteria (including Family Chloroflexaceae).
  • cyanobacteria The genera of cyanobacteria that may be used in various embodiments include Chamaesiphon, Chroococcus, Cyanobacterium, Cyanobium, Dactylococcopsis, Gloeobacter, Gloeocapsa, Gloeothece, Microcystis, Prochlorococcus, Prochloron, Synechococcus,
  • Cyanobacteria are photoautotrophs, able to use C0 2 as their sole carbon source and light as their energy source. Unlike certain other photosynthetic bacteria, cyanobacteria use the same photosynthetic pathway as eukaryotic cells such as algae and higher plants (the "C3" or "Calvin” cycle). Other photosynthetic bacteria use different light-harvesting pigments
  • the invention provides a genetically engineered photosynthetic bacteria ("host cell”) that expresses one or more heterologous enzyme and one or more heterologous transporter, wherein the transporter mediates the secretion of a product into the extracellular medium.
  • host cell a genetically engineered photosynthetic bacteria
  • the product secreted by the recombinant bacterium is a chiral compound.
  • chiral compounds include, without limitation, sugars and carboxylic acids, such as D-lactic acid and L-lactic acid.
  • sugars include lactose, galactose, glucose, fructose, a mixture of glucose and fructose, or sucrose.
  • chiral compounds include amino acids and vitamins.
  • the transporter mediates the secretion of a product whose synthesis is enhanced by the heterologous enzyme.
  • the heterologous enzymes can be of a type that catalyze a heterologous reaction that does not normally occur in the host organism, or can be an enzyme that is similar to an endogenous enzyme, but which catalyzes a desired reaction in a faster, more efficient, more specific, or otherwise preferable manner.
  • a heterologous enzyme is an enzyme that is not expressed naturally by the host cell.
  • heterologous enzymes include lactate dehydrogenase and invertase.
  • a "heterologous transporter" is a transporter that is not expressed naturally by the host cell.
  • a gene encoding an enzyme that participates in a pathway that leads to the synthesis of a compound is cloned into an expression vector for transformation into a photosynthetic bacterium.
  • the vector includes sequences that promote expression of the transgene of interest, such as a promoter, and may include an intron sequence, a sequence having a polyadenylation signal, etc.
  • the gene can be transformed into the cells such that it becomes operably linked to an endogenous promoter by homologous recombination or vector integration.
  • the heterologous genes may be "codon-optimized" for expression in an organism: the gene's nucleotide sequence has been altered with respect to the original nucleotide sequence such that one or more codons of the nucleotide sequence has been changed to a different codon that encodes the same amino acid, in which the new codon is used more frequently in genes of the host cell than the original codon.
  • the degeneracy of the genetic code provides that all amino acids except for methionine and tryptophan are encoded by more than one codon.
  • arginine, leucine, and serine are encoded by different six different codons; glycine, alanine, valine, threonine, and proline are encoded by four different codons.
  • Many organisms use certain codons to encode a particular amino acid more frequently than others. Without limiting any aspects of the invention to any particular mechanism, it is believed that some tRNAs for a given amino acid are more prevalent than others within a particular organism, and genes requiring a rare tRNA for translation of the encoded protein may be expressed at a low level due in part to a limiting amount of the rare tRNA.
  • a gene may be "codon-optimized” to change one or more codons to new codons ("preferred codons") that are among those used more frequently in the genes of the host organism (referred to as the "codon preference" of the organism).
  • a "codon-optimized” gene or nucleic acid molecule of the invention need not have every codon altered to conform to the codon preference of the intended host organism, nor is it required that altered codons of a "codon-optimized” gene or nucleic acid molecule be changed to the most prevalent codon used by the organism of interest.
  • a codon-optimized gene may have one or more codons changed to codons that are used more frequently that the original codon(s), whether or not they are used most frequently in the organism to encode a particular amino acid.
  • a variety of gene promoters that function in cyanobacteria can be utilized in expression vectors, including (a) the lac, tac, and trc promoters that are inducible by the addition of isopropyl ⁇ -D-l-thiogalactopyranoside (IPTG); (b) promoters that are naturally associated with transposon- or bacterial chromosome-borne antibiotic resistance genes (neomycin phosphotransferase, chloramphenicol acetyltrasferase, spectinomycin adenyltransferase, etc.); (c) promoters of various heterologous bacterial and native cyanobacterial genes; (d) promoters from viruses and phages; (e) salt-inducible promoters or other types of inducible promoters; and (f) synthetic promoters.
  • IPTG isopropyl ⁇ -D-l-thiogalactopyranoside
  • a heterologous enzyme at a certain point during the growth of the transgenic host to minimize any deleterious effects on the growth of the transgenic organism and/or to maximize yield of the desired product.
  • one or more exogenous genes introduced into the transgenic organism can be operably linked to an inducible promoter.
  • the promoter can be a lac promoter, a tet promoter (e.g., U.S. Patent No. 5,851,796), a hybrid promoter that includes either or both of portions of a tet or lac promoter, a hormone-responsive promoter (e.g., an ecdysone-responsive promoter, e.g., U.S. Patent No.
  • a metallothionine promoter U.S. Patent No. 6,410,828, or a promoter that can be responsive to a chemical such as, for example, salicylic acid, ethylene, thiamine, or BTH (U.S. Patent No. 5,689,044).
  • An inducible promoter can also be responsive to light or dark (U.S. Patents No. 5,750,385; No. 5,639,952) or temperature (U.S. Patent No. 5,447,858; Abe et al., 49 Plant Cell Physiol. 625 (2008); Shroda et al., 21 Plant J. 121 (2000)), or copper level.
  • the promoter sequences can be from any organism, provided that they are functional in the host organism.
  • Inducible promoters as used in the constructs of the present invention can use one or more portions or one or more domains of the aforementioned promoters or other inducible promoters fused to at least a portion of a different promoter that operates in the host organism to confer inducibility on a promoter that operates in the host species.
  • Promoters isolated from cyanobacteria that have been used successfully include secA (controlled by the redox state of the cell); rbc (Rubisco operon); psaAB (light regulated); psbA (light- inducible); and nirA (NH 3 /NO 3 regulated).
  • transcriptional terminators can be used for expression vector construction.
  • examples of possible terminators include, but are not limited to, psbA, psaAB, rbc, secA and T7 coat protein, as are known in the art.
  • a gene encoding an enzyme that participates in a pathway that leads to the synthesis of the chiral compound is cloned into an expression vector for transformation into a photosynthetic bacterium.
  • the vector includes sequences that promote expression of the transgene of interest, such as a promoter, an intron sequence, a sequence having a polyadenylation signal, etc.
  • the gene can be transformed into the cells such that it becomes operably linked to an endogenous promoter by homologous recombination or vector integration.
  • Transformation vectors can also include a selectable marker, such as but not limited to a drug resistance gene, an herbicide resistance gene, a metabolic enzyme or factor required for survival of the host (for example, an auxotrophic marker), etc.
  • a selectable marker such as but not limited to a drug resistance gene, an herbicide resistance gene, a metabolic enzyme or factor required for survival of the host (for example, an auxotrophic marker), etc.
  • Transformed cells can be optionally selected based upon the ability to grow in the presence of the antibiotic or other selectable marker under conditions in which cells lacking the resistance cassette or auxotrophic marker would not grow.
  • a non-selectable marker may be present on a vector, such as a gene encoding a fluorescent protein or enzyme that generates a detectable reaction product.
  • selectable or non- selectable markers can be provided on a separate construct, where both the gene-of-interest construct and the selectable marker construct are used together in transformation protocols, and selected transformants are analyzed for co-transformation of the construct that includes the gene- of-interest. See, e.g., Kindle, 87 PNAS 1228 (1990); Jakobiak et al., 155 Protist 381 (2004).
  • a vector is designed for integration of the heterologous nucleic acid sequence into the host genome.
  • Example vectors can be (a) targeted for integration into a bacterial chromosome by including flanking sequences that enable homologous recombination into the chromosome; (b) targeted for integration into endogenous host plasmids by including flanking sequences that enable homologous recombination into the endogenous plasmids; and/or (c) designed such that the expression vectors replicate within the chosen host.
  • Artificial chromosome vectors can also be used for the transformation of photosynthetic microorganisms when more than one gene that encodes an enzyme that participates in the synthesis and/or a transporter that enables secretion of a product as described herein is transformed into an organism.
  • the transporter is a member of the major facilitator superfamily.
  • This family of proteins includes the Glut family of glucose transport proteins in humans and other mammals, the HXT family of glucose transporters in yeast, the lacY gene product, the lldP lactate transporter of E. coli or another bacterium that performs anaerobic metabolism, a mammalian citric acid transport protein, an ion-coupled sucrose transporter such as cscB, and the glpT glycerol phosphate transporter.
  • the GLF protein of Z. mobilis is related to the glucose transporters from yeast and humans. These proteins have twelve membrane- spanning a-helices that form two rigid domains of six helices each. The subset of these proteins that includes the Glut, HXT and GLF proteins generally mediate transport of glucose, but their ability to transport other sugars such as fructose varies depending on the specific protein.
  • photosynthetic bacteria are engineered to synthesize a mixture of glucose and fructose. This is achieved by the expression of a heterologous invertase enzyme that catalyzes the breakdown of sucrose into glucose plus fructose. Synthesis of sucrose within the cell can be induced in certain cyanobacteria such as S. elongatus 7942 and
  • sucrose phosphate synthase and sucrose phosphate phosphatase expressed under the control of an inducible promoter with a different mode of regulation.
  • a heterologous hexose transporter is also expressed in the cyanobacteria, and this protein mediates the transport of glucose and fructose out of the cell.
  • a reduction-of-function mutation such as a deletion
  • this has the effect of reducing metabolism of glucose and/or fructose that is the desired product.
  • an additional gene that enhances production of UDP-glucose which is a precursor of sucrose, can be further expressed in engineered photosynthetic microbes.
  • the galU gene that encodes a UDP-glucose can be further expressed in engineered photosynthetic microbes.
  • pyrophosphorylase of a bacterium such as E. coli or a cyanobacterium can be used. Additional embodiments include over-expression of the genes that produce sucrose, which are sucrose phosphate synthase and sucrose phosphate phosphatase.
  • Another type of optimization relates to the type of promoter that is used for expression of the heterologous enzyme and/or transporter.
  • the enzyme is expressed from an inducible promoter.
  • the promoters used are selected from the lac operon promoter, a nitrogen- sensitive promoter, and a salt-inducible promoter.
  • the promoters for cyanobacterial nitrate reductase or nitrite reductase genes are repressed by ammonia and induced by nitrate; these are used as nitrogen-sensitive promoters.
  • NaCl-inducible promoters can be used for expression of the heterologous enzyme and/or transporter.
  • the NaCl-inducible promoters for the cyanobacterial sucrose phosphate synthase and sucrose phosphate phosphatase genes can be used for this purpose.
  • lactate dehydrogenase and a lactate transporter are expressed in a cyanobacterium.
  • the lactate dehydrogenase (IdhA) can be derived from any organism, such as from Gram-negative organism such as E. coli, from another cyanobacterium, or from a mammal such as a human.
  • lactate dehydrogenase of E. coli or any of a wide variety of other organisms is expressed in a cyanobacterium, and a lactate/H + co- transporter is also expressed.
  • the E. coli lactate/H + transporter illustrates many of the properties of this protein family.
  • the E. coli protein is encoded by the lldP gene and is able to transport L-lactate, D-lactate, and glycolate, making it useful for transporting a variety of molecules from engineered organisms.
  • This lactate transporter protein like the hexose transporter described above, is also in the major facilitator superfamily, of which members have twelve membrane-spanning ⁇ -helices, are thought to form two rigid domains of six helices each, and generally lack cleaved signal sequences.
  • lactate In the context of E. coli growing anaerobically, lactate is normally secreted as a waste product. The co-transport of a proton adds to the electrochemical gradient and export of lactate is thus an energy- generating step.
  • cyanobacteria When cyanobacteria are cultured, the pH of the medium often rises to pH 10 or pH 11 (Becking et al., 68 J. Geol. 243 (I960)).
  • Another aspect of the invention provides for recombinant photosynthetic bacteria, such as cyanobacteria, that express a heterologous transporter that is coupled to the pH gradient across a cell membrane.
  • genetically engineered photosynthetic bacteria such as cyanobacteria
  • an inorganic ion include, but not limited to, a proton, a sodium ion, a potassium ion, a chloride ion, and a hydroxide ion.
  • genetically engineered photosynthetic bacteria such as cyanobacteria
  • the invention provides genetically engineered photosynthetic bacteria, such as cyanobacteria, that express a heterologous transporter that co-transports a sodium ion with a metabolite.
  • the principle of coupling transport to the pH gradient is generalized.
  • Other bacteria express a number of transporters that, like the UdP protein, co- transport a given molecule with a proton or with a cation such as sodium that is coupled to the proton gradient.
  • Such other bacteria generally secrete protons into the extracellular space, resulting in acidification of the medium.
  • the secreted protons act as a form of stored energy, which is used when nutrient molecules are co-transported with a proton or sodium ion into the cell.
  • Most cyanobacteria tend to alkalinize their growth medium, which differentiates them from other bacteria.
  • the transporter can be a lactic acid/proton symporter, a sucrose proton symporter, a lactose/proton symporter, a dicarboxylic acid/proton symporter, an amino acid/proton symporter, a citrate/proton symporter, an amino acid/sodium ion symporter, or an amino acid/hydroxide ion antiporter.
  • Examples of additional proton-coupled transporters include the sucrose transporter CscB (Vadyvaloo et al., 358 J. Mol. Bio. 1051 (2006)), the lactose permease of E. coli encoded by the lacY gene (Guan et al., 35 Ann. Rev. Biophys. Biomol. Str. 67 (2006)), and the mammalian H + /peptide transporter PepTlH (Chen et al., 272 Biochem. Biophy. Res. Commn. 726 (2000)).
  • cyanobacteria start with light and C0 2 as feedstocks, the cost of production of any carbon-based molecule is a function of the number of photons needed to drive the synthesis of the molecule and the efficiency of the engineered pathway.
  • the cost of lactic acid is currently higher than the cost of sugar because sugar is used as a feedstock to produce lactic acid.
  • production of lactic acid by cyanobacteria can abolish this distinction, as the rates of production of sugars and lactic acid can become comparable.
  • Another aspect of the invention provides for the expression of a soluble
  • NAD(P)H transhydrogenase in photosynthetic microbes engineered to express lactate dehydrogenase or other enzymes that perform redox reactions.
  • it is often useful to eliminate, for example by mutation, any membrane-associated NAD(P) transhydrogenase.
  • one rationale is that most lactate dehydrogenase enzymes use NADH, rather than NADPH, as a substrate.
  • NADH rather than NADPH
  • NADPH is directly produced from electrons that emerge from Photosystem I via ferredoxin and ferredoxin-NADP reductase.
  • levels of NADPH generally exceed levels of NADH.
  • Expression of a soluble NAD(P)H transhydrogenase in sufficient quantities thus moves the balance of reducing equivalents between NAD and NADP toward equilibrium.
  • lactate and glucose transporters with twelve membrane- spanning regions represent a large class of such transporters, termed the major facilitatory superfamily. Marger et al., 18 Trends Biochem. Sci. 13 (1993). The lactate and glucose transporters, and many other proteins in this family, do not have signal sequences and the mechanism by which the proteins insert into the membrane is unknown. The results obtained here indicate that members of this superfamily can be co-expressed with appropriate enzymes, providing export of the
  • Rhodobacter in which a full length cDNA encoding yellow tail (Seriola quinqueradiata) growth hormone was cloned into an expression vector (under the lac promoter of E. coli) that contained a Rhodobacter-specific replicon.
  • the resulting plasmid was introduced by transformation into the marine purple non-sulfur photosynthetic bacterium Rhodobacter sp. strain NKPB0021.
  • the plasmid was maintained as an autonomous replicon and showed good stability in the absence of antibiotics. Burgess et al., 15 Biotech. Letts. I l l (1993).
  • Rhodobacter a photosynthetic variant was engineered to produce hydrogen in a light-independent manner by adding pyruvate lyase and formate lyase complex to a photosynthetic strain of Rhodobacter sphaeroides using E. coli conjugation.
  • U.S. Patent Appl. Pub. No. 2010/0003734 a photosynthetic variant was engineered to produce hydrogen in a light-independent manner by adding pyruvate lyase and formate lyase complex to a photosynthetic strain of Rhodobacter sphaeroides using E. coli conjugation.
  • a versatile Rhodobacter heterologous protein expression vector comprising a promoter nucleic acid sequence operable in a Rhodobacter, a nucleic acid sequence encoding an extended purification tag, a cloning cassette comprising a multiple cloning site and a selection marker has been described (WO 07/038746) and may be adapted for use in the present methods.
  • Another cloning system has been used to produce branched-chain alcohols via heterologous alcohol dehydrogenase in Synechococcus elongatus. More specifically, a construct combining Saccharomyces cerevisiae pyruvate decarboxylase gene and S. cerevisiae alcohol dehydrogenase gene cloned into S. elongatus PCC 7942. Also, a Lactococcus lactis KDCa gene was combined with S. cerevisiae ADH2 gene and transformed into S. elongatus cells as described by Golden and Sherman (158 J. Bacteriol. 36 (1984)). Additionally, a Lactococcus lactis KDCa gene was combined with S. cerevisiae ADH2 gene and transformed into S.
  • Synechocystis PCC 6803 Another cyanobacterium, Synechocystis PCC 6803, was transformed with plasmids encoding codon-modified S. cerevisiae PDC1 and ADH2 genes, as described by Zang et al. (45 J. Microbio. 241 (2007)); and the acetolactate synthase gene from Synechocystis sp. PCC 6803 was redesigned and cloned into wild-type Synechocystis as described by Zang et al., 2007. See U.S. Patent Appl. Pub. No. 2010/0151545.
  • PCC 7942 was constructed for the photosynthetic conversion of C0 2 to ethylene by inserting the ethylene-forming enzyme of Pseudomonas syringae. Sakai et al., 84 J. Ferment. Bioeng. 434 (1997). Synechococcus sp. PCC 7942 was also transformed with a recombinant plasmid harboring poly-(hydroxybutyrate) (PHB)-synthesizing genes from the bacterium Alcaligenes eutrophus. The transformant accumulated PHB in nitrogen-starved conditions, the PHB content held stable during a series of batch cultures, and the yield was increased by C0 2 -enrichment. Suzuki et al., 18 Biotech. Lett. 1047 (1996); Takahashi et al., 20 Biotech. Lett. 183 (1998).
  • PHB poly-(hydroxybutyrate)
  • Synechococcus R2 PCC7942 both as a plasmid-borne form and integrated into the chromosome.
  • a promoterless form of the lacZ gene was used as a reporter gene to make transcriptional fusions with cyanobacterial promoters using a shuttle vector system and also via a process of integration by homologous recombination.
  • Synechococcus R2 promoter-/acZ gene fusions were used to identify C0 2 -regulated promoters by quantitatively assessing the ⁇ -galactosidase activity under high and low C0 2 conditions, which detected several promoters induced under low C0 2 conditions. Scanlan et al., 90 Gene 43 (1990).
  • photosynthetic bacteria expressing a heterologous enzyme and a heterologous transporter as described herein can be further engineered to express one or more genes for enhancing production of hydrophilic products.
  • the additional genes encode enzymes for enhancing synthesis of intracellular precursors.
  • the galU gene from a bacterium such as E. coli can be further expressed in engineered photosynthetic microbes to increase intracellular sucrose, which is the precursor for production of glucose and fructose.
  • the udhA gene from a bacterium such as E.
  • NADH is the reducing substrate for lactate dehydrogenase required for conversion of pyruvate to lactate.
  • Alternative embodiments include reduction-of-function mutation, such as deletion of genes that causes a decrease in intracellular precursors or synthesized hydrophilic products via different cellular processes such as metabolism, degradation, post-modification, or co-binding with other products.
  • Still another aspect of the invention provides for a method for producing a hydrophilic product, the method comprising culturing a microbe according to the first aspect of the invention in the presence of light, C0 2 , appropriate minerals and water, and obtaining said the hydrophilic product from a supernatant of the cultured microbe.
  • the method further includes providing an inducing agent to induce the expression of at least one
  • heterologous gene expressed in photosynthetic microbe encodes an enzyme or a transporter.
  • the invention presented herein contemplates an attractive and economically feasible strategy for biofuel production using cyanobacteria in photobioreactors.
  • the invention provides a method for producing a hydrophilic product, the method comprising culturing a microbe according to the second aspect of the invention in the presence of light and C0 2 , appropriate minerals and water, and obtaining said the hydrophilic product from a supernatant of the cultured microbe.
  • the method further includes providing an inducing agent to induce the expression of at least one
  • heterologous gene expressed in photosynthetic microbe.
  • the heterologous gene encodes an enzyme, a transporter or both.
  • products can be purified from culture supernatants by standard techniques known to a skilled artisan. See, e.g., Ikeda, in 79 ADVANCES BIOCHEMICAL ENGIN. /BIOTECH.: MICROBIAL PROD, OF AMINO ACIDS, 1-35 (Faurie & T Subscribel, eds., Springer- Verlag, Berlin Heidelberg, Germany (2003)).
  • Sugars are purified by concentration and crystallization or simply obtained as sugar solutions with minor inorganic contaminants from the medium after filtration to remove bacteria.
  • Organic acids such as succinate or citrate are purified by acidification of the culture supernatant, followed by precipitation/crystallization of the organic acid, according to standard procedures well-known in the art.
  • a recombinant photosynthetic bacterium comprising at least one heterologous enzyme and at least one heterologous transporter, wherein said transporter mediates the secretion of a product whose synthesis is enhanced by said enzyme.
  • heterologous transporter encoded by a gene selected from the group consisting of: the Glut family of glucose transport proteins in humans and other mammals; the HXT family of glucose transporters in yeast; lacY; lldP lactate transporter of E. coli or another bacterium that performs anaerobic metabolism; a mammalian citric acid transport protein; the glpT glycerol phosphate transporter; and GLF of Z. mobilis.
  • a recombinant photosynthetic bacterium comprising at least one heterologous transporter, wherein said transporter mediates the symport or antiport of a product and an inorganic ion.
  • heterologous transporter encoded by a gene selected from the group consisting of: the sucrose transporter CscB; the lactose permease of E. coli encoded by the lacY gene; the mammalian H+/peptide transporter PepTlH; the glutamate transporter GltS and the lactate transporter UdP.
  • a method for producing a product comprising culturing a recombinant photosynthetic bacterium of any of paragraphs [0084] to [00100] in the presence of light and carbon dioxide, and obtaining the product from the supernatant of the cultured bacterium.
  • a method for producing a product comprising culturing a recombinant photosynthetic bacterium of any of paragraphs [00101] to [00111] in the presence of light and carbon dioxide, and obtaining the product from the supernatant of the cultured bacterium.
  • HM026754 IdhA in NS2
  • HM026755 MP in NS1
  • HM026756 glf NS1
  • HM026757 invA in NS2
  • HM026758 novel transgene in NS3
  • an engineered photosynthetic microbe that produces and secretes intracellular sugars, particularly glucose and fructose, was constructed as follows.
  • the specific microbe used in this Example was the cyanobacterium S. elongatus PCC7942 (Synechococcus), although a wide variety of cyanobacteria and other microbes could be used.
  • S. elongatus 7942 is a fresh water
  • S. elongatus 7942 that produce and secrete a mixture of glucose and fructose
  • two genes were introduced into S. elongatus 7942: (a) the Zymomonas mobilis invA gene encoding a soluble, cytoplasmic invertase (Yanase et al., 55 Agric. Biol. Chem. 1383 (1991)) to cleave NaCl-induced sucrose, and (b) the Z. mobilis glf gene encoding a glucrose- and fructose-facilitated diffusion transporter, which allows export of the sugars from the cell. Barnell et al., 172 J. Bacterid. 7227 (1990).
  • the GLF protein belongs to the major facilitator superfamily whose members have twelve transmembrane a- helical segments and generally lack cleaved signal sequences. DiMarco et al., 49 Appl.
  • Plasmid and strain construction The heterologous invA and glf genes were inserted into plasmid vectors that mediate integration into the S. elongatus 7942 genome by homologous recombination using neutral sites (Clerico et al., 362 Methods Mol. Biol. 155 (2007)) that can tolerate insertion with no phenotypic effects (Clerico et al., 362 Mets. Mol. Bio. 155 (2007); Mackey et al., 362 Mets. Mol. Bio. 15 (2007)).
  • Neutral site 1 (NS1) and NS2 are present in plasmids DS1321 and DS21, which confer spectinomycin and kanamycin resistance, respectively, and contain E. coli lad and an isopropyl- -D-thiogalactopyranoside (IPTG) -regulated trp-lac strong promoter. Diagrams of these vectors are shown in Figure 2.
  • the two genes were codon-optimized for expression in Synechococcus and were synthesized so that they contained a C-terminal His 6 tag. These genes were inserted into DS21 and DS1321 by using standard procedures.
  • the invA gene was inserted in NS 1 along with a spectinomycin-resistance marker and a lad gene; the invA gene was expressed from an E. coli trp/lac promoter and also encoded a C-terminal His 6 tag as an epitope for detection in western blots.
  • the glf gene was inserted in NS 2 along with a kanamycin-resistance marker and a lad gene; the glf gene was expressed from an E. coli lac promoter and also encoded a C-terminal His 6 tag as an epitope for detection in western blots. The sequences of these regions are shown in Figures 3 and 4, respectively.
  • the E. coli lad gene was also expressed in Synechococcus from its own promoter. Transformation of Synechococcus was performed as described previously. Clerico et al., 362 Mets. Mol. Bio. 155 (2007). Integration of vectors into neutral sites was verified by PCR to demonstrate the presence of appropriate novel chromosome-transgene junctions and the absence of uninserted sites.
  • Synechococcus strains expressing invA and glf were prepared from a culture that had been induced with 100 ⁇ IPTG for 3 days. Cell pellets were resuspended in invertase assay buffer and disrupted by sonication on ice for 2.5 sec followed by a 5-sec break, using a total sonication time of 5 min. Cell debris was removed by centrifugation. Invertase activity was measured as described (Yanase et al., 55 Agric. Biol. Chem. 1383 (1991)), using an assay mixture containing crude cell extracts in 100 mM sodium acetate buffer (pH 5) at 30°C. The reaction was started by addition of 150 mM sucrose.
  • the reaction was stopped by incubation at 100°C for 2 min.
  • the heat-denatured proteins were pelleted, and the glucose concentration in the supernatant was determined using a sucrose-glucose-fructose assay kit (Megazyme Intl., Wicklow, Ireland).
  • sucrose-glucose-fructose assay kit Megazyme Intl., Wicklow, Ireland.
  • the protein concentration was measured by performing a Bradford protein assay (Bio-Rad).
  • strains tested were a control strain carrying "empty vector" chromosomal insertions, strains carrying the glf gene or the invA gene with empty insertions corresponding to the other gene, and a strain carrying both the g//and invA genes. These strains are schematically shown in Figure 2.
  • Figure 6 shows that the Synechococcus strain expressing the invA and glf genes produced much larger amounts of glucose and fructose than the other strains, and the maximum total concentration was about 200 ⁇ when both invA and glf genes were expressed
  • Figure 7 shows the exponential growth rates of the four strains, and illustrates that the strain carrying neither transgene grew the fastest (0.80 ⁇ 0.002 per day), the strain earring the glf gene grew slightly more slowly (0.74 ⁇ 0.01 per day), and the strains carrying the invertase (0.61 ⁇ 0.01 per day) or the invertase gene and the glf gene grew the slowest (0.70 ⁇ 0.04 per day), but these growth rates were not dramatically different. This indicates that under the conditions employed, sugar production did not lead to a major diversion of the carbon flux.
  • Figure 8 shows the amount of sucrose produced by the engineered cyanobacteria. The levels of sucrose in the medium were low during the initial phase of the experiment.
  • Figure 8 shows that the extracellular sucrose was not detected in strains expressing invA only, or a combination of invA and glf and there were not significant differences between the wild-type and ⁇ //-expressing Synechococcus strains.
  • liquid cultures were inoculated with exponentially growing cells (e.g. cells from a 2-3 day old dense culture) at an optical density of 750nm (OD 750 ) of 0.05.
  • Sugar production was induced with 200 mM NaCl and 100 ⁇ IPTG. Alternative concentrations of NaCl for induction of sugar production range between 100 to 300 mM (in Example 2). Growth was monitored by measuring the OD 7 5 0 .
  • Sugar production was determined using a sucrose-D-fructose-D-glucose assay kit (catalogue number KSUFRG; Megazyme, Ltd., Bray, Ireland). Assays were performed using culture supematants prepared by centrifuging samples for 5 min at 21,130 x g. Assays were performed in triplicate, and standard deviations were determined.
  • Example 2 Production of hexoses as a function of salt concentration.
  • sugar yield was about 30% of the biomass produced by the corresponding empty vector control under identical growth conditions.
  • Example 3 Utilization of cyanobacterially secreted hexoses by a second bacterium.
  • FIGS 11A-11E Typical results are shown in Figures 11A-11E.
  • About 10 6 cells/ml of a prototrophic strain of E. coli that expressed the yellow fluorescent protein (YFP) were added to cultures of a g//+mvA-expressing strain of S. elongatus 7942 or a control strain at an OD 750 of 0.1.
  • YFP yellow fluorescent protein
  • 100 micromolar IPTG and 200 mM NaCl were also added to induce hexose secretion.
  • Figure 11A illustrates that both cyanobacterial strains continued to grow in the presence of the E. coli.
  • Figures 11B-11C illustrates that the E.
  • coculture of sugar- secreting cyanobacteria and a second engineered microbe can allow production of a desired product without a reduced-carbon feedstock in situations where synthesis of the product is incompatible with cyanobacterial metabolism.
  • the medium used was BG-11 medium with 100 ⁇ IPTG, 2.5 ⁇ g/ml kanamycin, 2 ⁇ g/ml spectinomycin, and 1 mg/ml NH 4 CI.
  • the E. coli strain carried a plasmid that was a hybrid of the pET47b (Kan), with a Spectinomycin-Resistance gene and a promoter driving expression of YFP.
  • the E. coli was also resistant to antibiotics used in the culture.
  • Growth of E. coli was monitored by plating serial dilutions on LB agar and measuring the YFP fluorescence of culture samples with a Victor 3V plate reader. For microscopic quantitation of Synechococcus and E. coli, bacteria in samples of the liquid coculture were visualized by red chlorophyll
  • Example 4 Production of lactic acid from a photo synthetic microbe.
  • Example 1 Using a similar approach to that was described in Example 1 for engineering of a photosynthetic bacterium to produce hexose sugars, a cyanobacterial strain was engineered to produce lactic acid. Specifically, lactate dehydrogenase IdhA from E. coli K12 (Plumbridge, 33 Mol. Microbio. 260 (1999)) was inserted into the DS21 plasmid, and the D,L-lactate transporter gene UdP from E. coli K12 (Nunez et al., 290 Biochem. Biophys. Res. Commun. 824 (2002)) was inserted into the DS1321 plasmid.
  • Diagrams of these plasmid vectors for expression of IdhA and UdP in S. elongatus are shown in Figures 12A-C, and the sequences of the inserted IdhA and UdP genes with their respective amino acid sequences are shown in Figures 13A-B and 14A-B, respectively.
  • E. coli IdhA gene encodes lactate dehydrogenase, which catalyzes the reduction of pyruvate to lactate (Plumbridge, 1999), and the E. coli UdP gene encodes a lactate transporter protein in the major facilitator superfamily. DiMarco et al., 1985; Snoep et al., 1994.
  • the LldP protein cotransports lactate with a proton (Nunez et al., 2002); during Synechococcus growth in BG-11 medium, when the culture reaches an OD 750 of 1, the pH of the medium is generally about pH 9, so lactate will be exported from the cell if it is produced intracellularly.
  • the schematic depiction of this engineering scheme is illustrated in Figure 15.
  • the IdhA and UdP were obtained by PCR amplification from E. coli DH5oc using oligonucleotides that resulted in molecules with an N-terminal His 6 tag.
  • expression of LdhA-His in Synechococcus was observed by western blotting, while LldP-Hise was not detected ( Figure 5B), perhaps due to low levels of expression combined with poor extraction from membranes. Expression of IdhA in Synechococcus resulted in increased levels of intracellular lactate.
  • Example 5 Enhanced production of lactic acid by additional engineering
  • lactic acid is enhanced by the expression of certain additional transgenes.
  • expression of a lactate operon from Lactococcus lactis leads to the production of L-lactic acid.
  • This enantiomer of D-lactic acid is also transported outside the cell by the UdP transporter.
  • This lactate operon includes the genes Lactate dehydrogenase, Pyruvate Kinase and Phosphofructokinase.
  • Photosynthetic microbes (e.g., S. elongatus 7942) produce NADPH as the major carrier of reducing equivalents, but lactate dehydrogenase uses NADH as its reducing substrate ( Figure 17A).
  • the engineered S. elongatus 7942 strain in Example 4 that expressed the IdhA and UdP genes of E. coli was further engineered to also express the udhA gene of E. coli, which encodes a soluble NADPH/NADH transhydrogenase (Boonstra et al, 181 J. Bacteriol. 1030 (1999)).
  • the IPTG- inducible lac promoter was used.
  • Synechococcus further expressing the udhA transhydrogenase indicates that on a per cell basis lactic acid by the IdhA, UdP, udhA strain was particularly high compared to its parent strain. Moreover, the initial rate of production of lactate in the strain expressing lactate dehydrogenase, the lactate transporter, and NADP/NAD transhydrogenase was about 54 mg/liter/day/OD 7 5 0 unit ( Figure 17B).
  • Plasmid and vector construction To express additional genes for pathway optimization, the udhA gene was introduced into a distinct plasmid integrating vector, termed pHNl-LacUV5, which was designed to integrate at a distinct NS 3 in the S. elongates 7942 genome ( Figures 19A-19B).
  • the vector expresses E. coli lad and encodes chloramphenicol resistance, mediates integration into the remnant of a cryptic prophage in the Synechococcus genome, and contains a lac operon promoter followed by a multiple-cloning site.
  • kanamycin 25 g/ml or 2.5 g/ml if in combination with other drugs
  • spectinomycin 25 g/ml or 2 g/ml if in combination with other drugs
  • chloramphenicol 12.5 ⁇ g/ml or 2.5 ⁇ g/ml if in combination with other drugs
  • Example 6 Enhanced production of hexose sugars by additional metabolic engineering
  • Sucrose is normally synthesized from fructose- 1 -phosphate and UDP-glucose, which are condensed to form sucrose phosphate that is then dephosphorylated to generate sucrose (Figure 19A).
  • UDP-glucose is synthesized by UDP-glucose phosphorylase, encoded by the E. coli galU gene. Marolda et al., 22 Mol. Microbiol 827 (1996).
  • proton symport proteins as transporters for the secretion of metabolites from cyanobacteria. Without wishing to be bound by theory, this is because cyanobacteria tend to alkalinize their growth medium so that when a metabolite is co-transported out of the cell with an H + ion, such transport is
  • a cyanobacterium such as Synechococcus elongatus PCC 7942 or Synechocystis species PCC 6803 is engineered to express the sucrose/H + symporter CscB.
  • the CscB protein is similar to LacY permease for lactose (Vadyvaloo et al., 358 J. Mol. Bio. 1051 (2006)); this protein is localized to the plasma membrane, has about twelve transmembrane a-helices, and key amino acids involved in cation co-transport are conserved between the LacY permease and the CscB protein.
  • the nuclei acid and amino acid coding sequences for the CscB protein of E. coli are given in Figures 20 A and 20B, respectively.
  • the CscB-encoding sequence is placed in an expression vector for S. elongatus PCC7942 in essentially the same manner as described above for the glf or lldP coding sequences as described above.
  • the promoter used is an inducible promoter such as a derivative of the E. coli lac promoter as described above, or a NaCl-inducible promoter. Alternatively, a constitutive promoter can be used.
  • the resulting CscB expression vector is introduced into S. elongatus PCC7942 by standard transformation procedures known to one of skill in the art.
  • the resulting S. elongatus PCC7942 strain expresses a functional CscB protein.
  • CscB protein evidence for functionality of the CscB protein is demonstrated, for example, as follows. Cyanobacteria expressing the CscB protein can grow very slowly in the absence of light when sucrose is present and the pH is buffered to about pH 7. Concomitant expression of invertase is optionally used to demonstrate this point. Alternatively, when the engineered cyanobacteria are induced to generate intracellular sucrose with NaCl, sucrose is found in the extracellular medium at levels significantly higher than the levels of sucrose in the medium of NaCl-induced, non-engineered S. elongatus PCC7942. For example, when a culture of CscB- expressing S.
  • sucrose phosphate synthase plus sucrose phosphate phosphatase are induced with 200 mM NaCl, levels of at least about 50 ⁇ sucrose accumulate in the extracellular medium within 2 days.
  • the amount of sucrose in the extracellular medium is less than one-half of the levels found in the cultures of the CscB-expressing recombinant S. elongatus.
  • Production of sucrose may be increased further by expression of enzymes involved in sucrose synthesis, such as sucrose phosphate synthase plus sucrose phosphate phosphatase, and/or such as UDP-glucose phosphorylase as described herein.
  • Example 8 Engineered synthesis and secretion of amino acids from a photo synthetic microbe.
  • Glutamate is a commercially important amino acid, being used as a food additive in the form monosodium glutamate (MSG).
  • MSG monosodium glutamate
  • a cyanobacterium such as Synechococcus elongatus PCC7942 or Synechocystis sp. PCC6803 is engineered to synthesize and secrete glutamate.
  • the glutamate transporter GltS promotes secretion of glutamate.
  • GltS co- transports glutamate and Na + ions.
  • Sodium and proton gradients are coupled by a Na + /H + antiporter, so that systems involving co-transport with Na + are indirectly coupled to the proton gradient.
  • metabolites produced by alkalinizing photosynthetic bacteria that are co- transported with Na + will accumulate outside of the cell, just as metabolites co-transported with H + will accumulate outside of the cell.
  • the nuclei acid and amino acid coding sequences for the GltS protein of E. coli are given in Figures 21 A and 21B.
  • the GltS-encoding sequence is placed in an expression vector for S. elongatus PCC7942 in essentially the same manner as described above for the glf or lldP coding sequences as described above.
  • the promoter used is an inducible promoter such as a derivative of the E. coli lac promoter as described above, or a NaCl-inducible promoter. Alternatively, a constitutive promoter can be used.
  • the resulting GltS expression vector is introduced into S. elongatus PCC7942 by standard transformation procedures known to one of skill in the art.
  • the resulting S. elongatus PCC7942 strain expresses a functional GltS protein.
  • GltS protein Evidence for functionality of the GltS protein is demonstrated, for example, as follows. When the recombinant cyanobacteria are induced to express GltS, glutamate
  • the amount of glutamate in the extracellular medium is less that one half of the levels found in the cultures of the recombinant, GltS-expressing S. elongatus.

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Abstract

L'invention porte sur de nouveaux procédés et compositions pour l’obtention de produits chimiques utiles à partir de bactéries photosynthétiques recombinantes telles que des cyanobactéries recombinantes. Les modes de réalisation de l'invention portent sur la modification génétique de bactéries photosynthétiques pour exprimer et secréter un produit d'intérêt tel que des sucres hexoses ou du lactate. Plus particulièrement, les cyanobactéries recombinantes expriment des gènes hétérologues pour à la fois l'expression et la sécrétion du produit. Dans certains modes de réalisation, les cyanobactéries recombinantes expriment des gènes hétérologues pour la sécrétion de produits couplée à un gradient de pH à travers une membrane cellulaire. Les sucres, tels que le saccharose, le glucose et le fructose et d'autres produits chimiques utiles, tels que l'acide lactique, sont obtenus par l'invention.
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US8227237B2 (en) 2008-03-03 2012-07-24 Joule Unlimited Technologies, Inc. Engineered CO2 fixing microorganisms producing carbon-based products of interest
US8835137B2 (en) 2008-12-23 2014-09-16 Matrix Genetics, Llc Modified photosynthetic microorganisms with reduced glycogen and their use in producing carbon-based products
US8980613B2 (en) 2010-04-06 2015-03-17 Matrix Genetics, Llc Modified photosynthetic microorganisms for producing lipids
WO2016105483A1 (fr) * 2014-12-23 2016-06-30 Algenol Biotech LLC Procédés pour augmenter la stabilité de la production de composés dans des cellules hôtes microbiennes
US10138489B2 (en) 2016-10-20 2018-11-27 Algenol Biotech LLC Cyanobacterial strains capable of utilizing phosphite
WO2023001294A1 (fr) * 2021-07-22 2023-01-26 深圳先进技术研究院 Procédé de production de glucose et de ses dérivés au moyen d'une biotransformation à l'aide de levure recombinée
CN115927131A (zh) * 2022-04-28 2023-04-07 中国科学院青岛生物能源与过程研究所 可分泌和高产果糖的聚球藻yd03及其制备方法
WO2024098042A3 (fr) * 2022-11-04 2024-08-02 The Regents Of The University Of California Plate-forme de sélection basée sur la croissance pour enzymes oxydantes à cofacteur non canoniques
US12252513B2 (en) 2018-07-16 2025-03-18 Lumen Bioscience, Inc. Thermostable phycobiliproteins produced from recombinant arthrospira
US12447202B2 (en) 2018-05-17 2025-10-21 Lumen Bioscience, Inc. Arthrospira platensis oral vaccine delivery platform
US12503682B2 (en) 2019-07-03 2025-12-23 Lumen Bioscience, Inc. Arthrospira platensis non-parenteral therapeutic delivery platform

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US8227237B2 (en) 2008-03-03 2012-07-24 Joule Unlimited Technologies, Inc. Engineered CO2 fixing microorganisms producing carbon-based products of interest
US8835137B2 (en) 2008-12-23 2014-09-16 Matrix Genetics, Llc Modified photosynthetic microorganisms with reduced glycogen and their use in producing carbon-based products
US8980613B2 (en) 2010-04-06 2015-03-17 Matrix Genetics, Llc Modified photosynthetic microorganisms for producing lipids
WO2016105483A1 (fr) * 2014-12-23 2016-06-30 Algenol Biotech LLC Procédés pour augmenter la stabilité de la production de composés dans des cellules hôtes microbiennes
US10174329B2 (en) 2014-12-23 2019-01-08 Algenol Biotech LLC Methods for increasing the stability of production of compounds in microbial host cells
US10138489B2 (en) 2016-10-20 2018-11-27 Algenol Biotech LLC Cyanobacterial strains capable of utilizing phosphite
US12447202B2 (en) 2018-05-17 2025-10-21 Lumen Bioscience, Inc. Arthrospira platensis oral vaccine delivery platform
US12252513B2 (en) 2018-07-16 2025-03-18 Lumen Bioscience, Inc. Thermostable phycobiliproteins produced from recombinant arthrospira
US12503682B2 (en) 2019-07-03 2025-12-23 Lumen Bioscience, Inc. Arthrospira platensis non-parenteral therapeutic delivery platform
WO2023001294A1 (fr) * 2021-07-22 2023-01-26 深圳先进技术研究院 Procédé de production de glucose et de ses dérivés au moyen d'une biotransformation à l'aide de levure recombinée
CN115927131A (zh) * 2022-04-28 2023-04-07 中国科学院青岛生物能源与过程研究所 可分泌和高产果糖的聚球藻yd03及其制备方法
WO2024098042A3 (fr) * 2022-11-04 2024-08-02 The Regents Of The University Of California Plate-forme de sélection basée sur la croissance pour enzymes oxydantes à cofacteur non canoniques

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